The long-term objective of this research is to study the effect of altered gene transcription on drug-resistance in Trichomonas vaginalis - a common etiologic agent of vaginitis. Trichomoniasis is treated with metronidazole, a drug that is administered in an inactive form and is rendered cytotoxic within the parasite. We have observed that transcription of a gene that encodes a protein necessary for metronidazole activation is invariably reduced in T. vaginalis drug- resistant strains. To identify transcription defects that may give rise to drug-resistance, the basic mechanisms governing transcriptional regulation must first be defined. This proposal focuses on identifying transcriptional regulatory elements and characterizing the RNA polymerase that transcribes protein-coding genes in T. vaginalis. In addition to providing insight into gene expression and ultimately drug resistance, these studies may also reveal transcriptional properties of this parasite that distinguish it from the human host, providing possible therapeutic targets. Our previous studies on transcription in trichomonads have shown that (1) the transcription of protein-coding genes is relatively insensitive to alpha-amanitin and (2) all examined protein-coding genes contain a highly conserved 13 nucleotide motif that contains the start of transcription. This conserved motif is structurally and functionally similar to initiator (Inr) elements found in higher eukaryotic genes. We propose to extend these observations by: (1) analyzing the RNA polymerase (RNAP) that transcribes protein-coding genes in T. vaginalis by characterizing genes that encode the large subunit of RNAP and comparing these genes with their counterparts in eubacteria, archaebacteria and other eukaryotes (2) complementing yeast that are deficient in the large subunit of RNAP II with the T. vaginalis large subunit gene to test if complementation confers alpha-amanitin insensitive protein-coding gene transcription (3) characterizing T. vaginalis Inr elements by examining additional genes and utilizing our newly established transient transfection assays to study the role of Inr elements in vivo and (4) isolating proteins that interact with Inr elements and testing the effect of these proteins on transcription using nuclear run-on assays. These studies will define factors that play a pivotal role in transcription in trichomonads and will allow further investigation of alterations in transcription that may lead to drug resistance.